This invention relates to novel formulations of nucleic acid pharmaceutical products, specifically formulations of nucleic acid vaccine products and nucleic acid gene therapy products. The formulations of the disclosure stabilize the conformation of DNA pharmaceutical products. The vaccines, when introduced directly into muscle cells, induce the production of immune responses which specifically recognize human influenza virus.
During storage as a pharmaceutical entity, DNA plasmid vaccines undergo a physicochemical change in which the supercoiled plasmid converts to the open circular and linear form. A variety of storage conditions (low pH, high temperature, low ionic strength) can accelerate this process. In this invention, the removal and/or chelation of trace metal ions (with succinic or malic acid, or with chelators containing multiple phosphate ligands) from the DNA plasmid solution, from the formulation buffers or from the vials and closures, stabilizes the DNA plasmid from this degradation pathway during storage. In addition, non-reducing free radical scavengers are required to prevent damage of the DNA plasmid from free radical production that may still occur, even in apparently demetalated solutions. Furthermore, the buffer type, pH, salt concentration, light exposure, as well as the type of sterilization process used to prepare the vials, all must be controlled in the formulation to optimize the stability of the DNA vaccine. Lyophilization of the DNA vaccine in the presence of the appropriate formulation excipients can also be performed to stabilize the plasmid during storage.
From the scientific literature, the chain scission reaction causing conversion of supercoiled to open circular to linear DNA plasmid would be expected to occur via two different chemical mechanisms (since these preparations of highly purified DNA do not contain nucleases): (1) depurination followed by β-elimination and/or (2) free radical oxidation. Although removal of trace metal ions would be expected to suppress the free radical oxidation mechanism of DNA chain scission, surprisingly, our results indicate that the removal or chelation of trace metal ions from the DNA containing solution, stabilizes the DNA against both mechanisms of degradation, as judged by comparison of our stability data with the published rates of depurination and β-elimination (see Lindahl et al., 1972, Biochemistry 19: 3610-3618; Lindahl et al., 1972, Biochemistry 19: 3618-3623). Based on these and other published reports, the removal of trace metal ions would not be expected to have a significant effect on the rates of depurination or β-elimination. Therefore, the increase in DNA stability resulting from the removal of trace metal ions is much larger than expected, and cannot be explained on the basis of the published rate constants for depurination and β-elimination.
In addition, our data indicates that specific chelating agents such as inositol hexaphosphate, tripolyphosphate, succinic and malic is acid, increase the stability of plasmid DNA in storage, while other commonly used chelating agents such as EDTA, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA) provide no significant enhancement of stability. These results also suggest that any chelating agent with multiple phosphate ligands (for example, polyphosphoric acid) will enhance DNA stability. It is not clear from the published literature, however, why inositol hexaphosphate stabilizes DNA, but EDDHA, desferal and DTPA do not. Since the published literature suggests that all four of these chelators inhibit the production of hydroxyl radicals catalyzed by iron, it was expected that all of these reagents would provide enhanced DNA stability (by chelating trace metal ions and inhibiting the production of free radicals), but this was not observed. Moreover, the literature reports that both EDTA and ATP support metal ion catalyzed hydroxyl radical production, but we have observed that tripolyphosphate (the metal binding moiety of ATP) enhances DNA stability while EDTA does not. Therefore, the protective effects of the metal ion chelators do not appear to be directly correlated with their ability to support the production of hydroxyl radicals. The identification of the appropriate chelators to stabilize DNA formulations will require empirical testing as described in this work.
In addition to the removal and/or chelation of trace metal ions, the use of non-reducing free radical scavengers is important for stabilizing DNA formulations during storage. Our results indicate that ethanol, methionine, glycerol and dimethyl sulfoxide enhance DNA stability, suggesting that their protective effect is due to the scavenging of free radicals. Furthermore, our results indicate that scavengers capable of serving as reducing agents, such as ascorbic acid, greatly accelerate DNA degradation, presumably by acting as a reducing agent to keep trace metal ions in their reduced (most damaging) state. Our results also indicate that several scavengers expected to stabilize DNA (based on known rate constants with hydroxyl radical) unexpectedly accelerated DNA degradation, or provided no increase in stability. For example, pentoxifylline and para-aminobenzoic acid are hydroxyl radical scavengers with large rate constants for hydroxyl radicals (k=1.1×1010 M−1 s−1; see Freitas and Filipe, 1995, Biol. Trace Elem. Res. 47: 307-311; Hu et al., 1995, J. Nutr. Biochem. 6: 504-508), yet pentoxifylline did not enhance stability and p-aminobenzoic acid actually accelerated DNA degradation. Because of these results, the empirical screening of a number of free radical scavengers has been the most effective means of identifying useful compounds.
To maximize DNA stability in a pharmaceutical formulation, the type of buffer, salt concentration, pH, light exposure as well as the type of sterilization process used to prepare the vials are all important parameters that must be controlled in the formulation to further optimize the stability. Furthermore, lyophilization of the DNA vaccine with appropriate formulation excipients can also be performed enhance DNA stability, presumably by reducing molecular motion via dehydration. Therefore, our data suggest that the formulation that will provide the highest stability of the DNA vaccine will be one that includes a demetalated solution containing a buffer (phosphate or bicarbonate) with a pH in the range of 7-8, a salt NaCl, KCl or LiCl) in the range of 100-200 mM, a metal ion chelator (succinate, malate, inositol hexaphosphate, tripolyphosphate or polyphosphoric acid), a non-reducing free radical scavenger (ethanol, glycerol, methionine or dimethyl sulfoxide) and the highest appropriate DNA concentration in a sterile glass vial, packaged to protect the highly purified, nuclease free DNA from light.
The instant formulations and methods are exemplified with a DNA vaccine against influenza. Nothing in this disclosure should be construed as limiting the formulations and methods to the specific DNA vaccine.
Influenza is an acute febrile illness caused by infection of the respiratory tract with influenza A or B virus. Outbreaks of influenza occur worldwide nearly every year with periodic epidemics or pandemics. Influenza can cause significant systemic symptoms, severe illness (such as viral pneumonia) requiring hospitalization, and complications such as secondary bacterial pneumonia. Recent U.S. epidemics are thought to have resulted in >10,000 (up to 40,000) excess deaths per year and 5,000-10,000 deaths per year in non-epidemic years. The best strategy for prevention of the morbidity and mortality associated with influenza is vaccination. The current licensed vaccines are derived from virus grown in eggs, then inactivated, and include three virus strains (two A strains and one B strain). Three types of vaccines are available: whole-virus, subvirion, and purified surface antigen. Only the latter two are used in children because of increased febrile responses with the whole-virus vaccine. Children under the age of 9 require two immunizations, while adults require only a single injection. However, it has been suggested [see Medical Letter 32:89-90, Sep. 17, 1993] that “patients vaccinated early in the autumn might benefit from a second dose in the winter or early spring,” due to the observations that in some elderly patients, the antibody titers following vaccination may decline to less-than-protective levels within four months or less. These vaccines are reformulated every year by predicting which recent viral strains will clinically circulate and evaluating which new virulent strain is expected to be predominant in the coming flu season. Revaccination is recommended annually.
The limitations of the licensed vaccine include the following:
1) Antigenic variation, particularly in A strains of influenza, results in viruses that are not neutralized by antibodies generated by a previous vaccine (or previous infection). New strains arise by point mutations (antigenic drift) and by reassortment (antigenic shift) of the genes encoding the surface glycoproteins (hemagglutinin [HA] and neuraminidase), while the internal proteins are highly conserved among drifted and shifted strains. Immunization elicits “homologous” strain-specific antibody-mediated immunity, not “heterologous” group-common immunity based on cell-mediated immunity.
2) Even if the predominant, circulating strains of influenza virus do not shift or drift significantly from one year to the next, immunization must be given each year because antibody titers decline. Although hemagglutination-inhibiting (HI) and neutralizing antibodies are reported by some to persist for months to years with a subsequent gradual decline, the Advisory Committee on Immunization Practices cites the decline in antibody titers in the year following vaccination as a reason for annual immunization even when there has been no major drift or shift. (HI antibodies inhibit the ability of influenza virus to agglutinate red blood cells. Like neutralizing antibodies, they are primarily directed against the HA antigen. Hemagglutination inhibition tests are easier and less expensive to perform than neutralization assays are, and thus are often used as a means to assess the ability of antibodies raised against one strains of influenza to react to a different strain). As mentioned above, The Medical Letter suggests that certain high-risk, older individuals should be vaccinated twice in one season due to short-lived protective antibody titers.
3) The effectiveness of the vaccine is suboptimal. Development of the next season's vaccine relies upon predicting the upcoming circulating strains (via sentinel sampling in Asia), which is inexact and can result in a poor match between strains used for the vaccine and those that actually circulate in the field. Moreover, as occurred during the 1992-1993 flu season, a new H3N2 strain (A/Beijing/92) became clinically apparent during the latter phase of the flu season. This prompted a change in the composition of the 1993-1994 vaccine, due to poor cross-reactivity with A/Beijing/92 of the antibody induced by the earlier H3N2 strain (A/Beijing/89) due to antigenic shift. However, due to the length of time needed to make and formulate the current licensed vaccine, the new vaccine strain could not be introduced during the 1992-1993 season despite the evidence for poor protection from the existing vaccine and the increased virulence of the new circulating H3N2 strain.
Characteristics of an Ideal Universal Influenza Vaccine include the following:
1) Generation of group-common (heterologous) protection.
2) Increased breadth of antibody response. Because CTL are thought to play a role in recovery from disease, a vaccine based solely upon a CTL response would be expected to shorten the duration of illness (potentially to the point of rendering illness subclinical), but it would not prevent illness completely.
3) Increased duration of antibody responses. Because one of the very groups that is at highest risk for the morbidity and mortality of influenza infection (elderly) is also the group in whom protective antibody titers may decline too rapidly for annual immunization to be effective, an improved vaccine should generate protective titers of antibody that persist longer.
Intramuscular inoculation of polynucleotide constructs, i.e., DNA plasmids encoding proteins have been shown to result in the in situ generation of the protein in muscle cells. By using cDNA plasmids encoding viral proteins, both antibody and CTL responses were generated, providing homologous and heterologous protection against subsequent challenge with either the homologous or cross-strain protection, respectively. Each of these types of immune responses offers a potential advantage over existing vaccination strategies. The use of PNVs (polynucletide vaccines) to generate antibodies may result in an increased duration of the antibody responses as well as the provision of an antigen that can have both the exact sequence of the clinically circulating strain of virus as well as the proper post-translational modifications and conformation of the native protein (vs. a recombinant protein). The generation of CTL responses by this means offers the benefits of cross-strain protection without the use of a live potentially pathogenic vector or attenuated virus.
Therefore, this invention contemplates any of the known methods for introducing nucleic acids into living tissue to induce expression of proteins. This invention provides a method for introducing viral proteins into the antigen processing pathway to generate virus-specific CTLs. Thus, the need for specific therapeutic agents capable of eliciting desired prophylactic immune responses against viral pathogens is met for influenza virus by this invention. Of particular importance in this therapeutic approach is the ability to induce T-cell immune responses which can prevent infections even of virus strains which are heterologous to the strain from which the antigen gene was obtained. Therefore, this invention provides DNA constructs encoding viral proteins of the human influenza virus nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NM), matrix (M), nonstructural (NS), polymerase (PB1 and PB2=basic polymerases 1 and 2; PA=acidic polymerase) or any of the other influenza genes which encode products which generate specific CTLs.